US11898146B2ActiveUtilityA1

Aptamers against Clostridium difficile, compositions comprising aptamers against Clostridium difficile and methods of using the same

87
Assignee: LIV PROCESS INCPriority: Dec 3, 2020Filed: Dec 3, 2020Granted: Feb 13, 2024
Est. expiryDec 3, 2040(~14.4 yrs left)· nominal 20-yr term from priority
Inventors:Ronald J. ShannonGregory Allen Penner
C12N 15/115G01N 21/6428G01N 33/56911C12N 2310/16C12N 2310/531G01N 2021/6439G01N 33/5308G01N 2333/33G01N 33/542C12N 2310/3517C12N 2320/31C12N 2310/151
87
PatentIndex Score
2
Cited by
47
References
26
Claims

Abstract

Compositions comprising optimized aptamers capable of specifically binding to a surface protein of Clostridium difficile spore are provided. A method for detecting, enriching, separating, and/or isolating Clostridium difficile spores is provided.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A method of visualizing  Clostridium difficile  spores on a surface, comprising:
 contacting a surface with a liquid composition comprising (a) at least one aptamer conjugated to a fluorophore, wherein the at least one aptamer has a stem-loop structure having a specific binding affinity for a surface protein of  Clostridium difficile  spore, wherein the at least one aptamer comprises a nucleic acid sequence having at least 90% identity with any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 43-55 wherein the surface protein is a spore coat surface protein or an exosporium layer protein; and (b) graphene oxide, wherein fluorophore is quenched by the association with the graphene oxide; and 
 visualizing the presence or absence of  Clostridium difficile  spores on the surface, wherein fluorescence is visible when the at least one aptamer is bound to the surface protein of  Clostridium difficile  spores. 
 
     
     
       2. The method of  claim 1 , wherein when the at least one aptamer is not bound to the surface protein, the fluorophore is quenched and wherein when the aptamer binds to the surface protein, the fluorophore is not quenched. 
     
     
       3. The method of  claim 1 , wherein the surface protein is CdeM, or CotE Chitinase. 
     
     
       4. The method of  claim 1 , wherein the at least one aptamer comprises a nucleic acid sequence that selectively binds to surface protein CdeM of the  Clostridium difficile  spores, the aptamer having a stem-loop structure comprising Loop(3)/Stem(4f)/Loop(5)/Stem(4r)/Loop(5)/Stem(2f)/Loop(1)/Stem(2f)/Loop(1)/Stem(5 f)/Loop(5)/Stem(5r)/Loop(1)/Stem(2r)/Loop(1)/Stem(2r)/Loop(3). 
     
     
       5. The method of  claim 1 , wherein the composition comprises two or more aptamers having a binding affinity to two or more epitopes of a surface protein of the  Clostridium difficile  spores or to two or more surface proteins of the  Clostridium difficile  spores. 
     
     
       6. The method of  claim 1 , wherein the graphene oxide is in the form of nanoparticles. 
     
     
       7. The method of  claim 1 , wherein the fluorophore emits at a wavelength of between about 510 nm and about 520 nm. 
     
     
       8. The method of  claim 1 , further comprising illuminating the surface with a light source. 
     
     
       9. The method of  claim 8 , wherein light from the light source has a predetermined wavelength, and the predetermined wavelength is different than a wavelength of light emitted by the fluorophore of the aptamer conjugate. 
     
     
       10. The method of  claim 8 , wherein the light source is configured to produce light at a wavelength of between about 485 nm and about 515 nm. 
     
     
       11. The method of  claim 8 , further comprising filtering the light produced by the light source such that light at a wavelength emitted by the fluorophore is visually detected. 
     
     
       12. The method of  claim 8 , comprising passing the light produced from the light source through a bandpass filter such that light at a wavelength emitted by the fluorophore is visually detected. 
     
     
       13. The method of  claim 8 , comprising passing the light produced from the light source through a circular polarizing filter such that light at a wavelength emitted by the fluorophore is visually detected. 
     
     
       14. The method of  claim 1 , wherein the contacting comprises spraying. 
     
     
       15. A method of visualizing  Clostridium difficile  spores on a surface, comprising:
 contacting a surface with a liquid composition comprising (a) at least one aptamer conjugated to a fluorophore, wherein the at least one aptamer has a stem-loop structure having a specific binding affinity for a surface protein of  Clostridium difficile  spore, wherein the at least one aptamer comprises a nucleic acid sequence as set forth in SEQ ID NO: 55, wherein the surface protein is a spore coat surface protein or an exosporium layer protein; and (b) graphene oxide, wherein fluorophore is quenched by the association with the graphene oxide; and 
 visualizing the presence or absence of  Clostridium difficile  spores on the surface, wherein fluorescence is visible when the at least one aptamer is bound to the surface protein of  Clostridium difficile  spores. 
 
     
     
       16. The method of  claim 15 , wherein when the at least one aptamer is not bound to the surface protein, the fluorophore is quenched and wherein when the aptamer binds to the surface protein, the fluorophore is not quenched. 
     
     
       17. The method of  claim 15 , wherein the at least one aptamer comprises a nucleic acid sequence that selectively binds to surface protein CdeM of the  Clostridium difficile  spores, the aptamer having a stem-loop structure comprising Loop(3)/Stem(4f)/Loop(5)/Stem(4r)/Loop(5)/Stem(2f)/Loop(1)/Stem(2f)/Loop(1)/Stem(5 f)/Loop(5)/Stem(5r)/Loop(1)/Stem(2r)/Loop(1)/Stem(2r)/Loop(3). 
     
     
       18. The method of  claim 15 , wherein the composition comprises two or more aptamers having a binding affinity to two or more epitopes of a surface protein of the  Clostridium difficile  spores or to two or more surface proteins of the  Clostridium difficile  spores. 
     
     
       19. The method of  claim 15 , wherein the graphene oxide is in the form of nanoparticles. 
     
     
       20. The method of  claim 15 , wherein the fluorophore emits at a wavelength of between about 510 nm and about 520 nm. 
     
     
       21. The method of  claim 15 , further comprising illuminating the surface with a light source. 
     
     
       22. The method of  claim 21 , wherein light from the light source has a predetermined wavelength, and the predetermined wavelength is different than a wavelength of light emitted by the fluorophore of the aptamer conjugate. 
     
     
       23. The method of  claim 21 , wherein the light source is configured to produce light at a wavelength of between about 485 nm and about 515 nm. 
     
     
       24. The method of  claim 21 , further comprising filtering the light produced by the light source such that light at a wavelength emitted by the fluorophore is visually detected. 
     
     
       25. The method of  claim 21 , comprising passing the light produced from the light source through a bandpass filter such that light at a wavelength emitted by the fluorophore is visually detected. 
     
     
       26. The method of  claim 21 , comprising passing the light produced from the light source through a circular polarizing filter such that light at a wavelength emitted by the fluorophore is visually detected.

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